Preservation of leucocytes in blood



July 18, 1950 H. BROWN ETAL PRESERVATION'OF LEUCOCYTES IN BLODD Filed Oct. 12, 1948 'INVENTORS He r ma Bron/72 1W2 yer- 6' Patented July 18, 1950 PRESERVATION OF LEUCOCYTES IN BLOOD- Herman Brown, Philadelphia, and Meyer Samson, Melrose Park, Pa.; said Brown assignor to said Samson Application October 12, 1948, Serial No. 54,106

15 Claims.

This invention relates to the preservation of blood and has for one of its primary objects the preservation of blood for the purpose of making possible accurate determinations of its hemoglobin, erythrocyte and leucocyte contents in stored specimens. Other objects will become apparent from the following description thereof and accompanying drawings, in which like reference numerals indicate like parts and in which Figure 1 represents an enlarged perspective view of a specimen tube exemplifying one embodiment of that phase of the present invention which may be regarded as the container phase of the present invention; shown on an enlarged scale which is approximately five times actual size, and

Figure 2;represents a cross-sectional view on line 22 of Figure 1. r

It has been possible in the past to perform hemoglobin determinations and erythrocyte counts on stored samples of blood, provided the specimen was taken with due precaution to avoid bacterial contamination and the anticoagulants used were not hemolytic in nature. Delayed determination of the leucocyte content of blood has not, however, been practical in the past due to the fact that room temperatures or the higher ones prevailing under conditions of public transport cause rapid breakdown and eventual disappearance of these elements of the blood.

The disappearance of the leucocytes from the blood takes place by a process known as leucolysis. This disappearance is accelerated at body temperatures, which are higher than ordinary room temperatures. In common with other cells in nature, leucocytes contain within their protoplasm enzymes which cause degradation of the cellular content after death of the cell. This general phenomenon, known as cellular autolysis, is termed leucolysis, when it refers to leucocytes. The exact nature of the enzymes concerned is tionis that the enzymes known as .proteinases and peptidases are the ones mostly responsible for this particular type of autolysis.

The customary hematologic examination used. as an aid to medical practice is known as a com plete blood count. It entails: a determination of the hemoglobin by colorimetric means; a count of the erythrocytes by microscopic examination of accurately diluted specimens, or by turbidimetric tests yielding equivalent values; a count of the leucocytes by similar microscopic means; and a high powered microscopic examination of a stained unicellular smear of the blood. for the purpose of difierentiating the leucocytes into the various types; and of examination of the erythro-v cytes morphologically and cytologically for deviations from the norm.

Because of the above mentioned leucolysis it has not been possible in the past to make correct leucocyte counts on blood which had been taken more than a few hours and kept at normal room temperatures previous to examination in the laboratory. For this reason it has been a long sought for objective in the clinical laboratory field to be able to perform such examinations on specimens taken from patients who are far from the examining laboratory or who may for other reasons find it impossible to come to the laboratory.

Our discovery relates to the inhibition of leucolysis in a degree sufficient to make delayed enumeration of the leucocytes possible and also otherwise to utilize the preservative effects and advantages of such inhibition.

We have discovered that leucolysis may be inhibited by the addition to the fresh blood of a small amount of soluble salts either of orthohydroxy benzoic acid or the derivatives thereof wherein the hydrogen atom of the hydroxyl group has been substituted by other groups, as for exnot clearly understood. The present presumpample, ortho acetoxy benzoic acid. Used for the purposes described, in conjunction with anticoagulants commonly used in hematologic work. such as oxalates, citrates, pyrophosphates, heparin and the like, the anti-leucolytic substances discovered by us in no way cause hemolysis of the red cells present in the blood. A correct erythrocyte' count and hemoglobin determination are therefore also possible on the stored sample.

For transmitting a specimen of blood from the bedside or from a physicians office for a blood count, We have found it convenient to use a small specimen tube 3 on the inner wall 4 of which there is coated or deposited, either uniformly or as a blob or zone 5, a small quantity of an appropriate anticoagulant and one of the antileucolytic substances of this discovery.

The freshly drawn blood may be transferred to the tube 3, prepared in the foregoing fashion, which is then closed, by any suitable closure 6, shaken to dissolve the preservative, and forwarded to the laboratory wherein the blood count is to be made. With the blood so treated there is no need for haste in making the blood count as the leucocytes as well as the hemoglobin and the erythrocytes are preserved.

If desired, a horizontal and more or less encircling marker-line 1 may be applied to the outside (or inside) of the tube 3, a suitable distance below the top thereof, to mark off the level (below the top) to which the tube should be filled with the blood to be examined (hematologically). Thus, for example, a glass or transparent plastic tube 3 whose total interior has a volume of two cubic centimeters (2 cc.) has a line I (printed, painted, etched or: otherwise applied thereto) about a quarter n) of the way down from its top, so as to mark the level of one and one-half.

cubic centimeters (l /2 cc.) from the bottom of the tube 3. The amount of anticoagulant and anti- 1 leucolytic substance 5 applied to the inner wall 4 of the tube 3 is such that when dissolved in 1% 1 cc. to 2 cc. of blood it will give a concentration of anti-leucolytic substance within the range of concentration hereinbelow indicated; it. being. I desirable to fill the tube 3 only to the .line I in order to afiord some unoccupied space within the tube 3, within which the blood may better be shaken up so as quickly to dissolve the anti coagulant and anti-leucolytic substance 5. How- 4 ever, the amount of anti-coagulant and antileucolytic substance is such that it will also be sufiicient to give the below-indicated concentration in a, volume of blood which may fill the tube 3 in its entirety.

We have found that a 0.01 molar concentration of soluble salts. of salicylic acid andacetyl salicylic acid is effective in inhibiting leucolysis. Much larger quantities up to about 0.08 molar concentration of the inhibitor may be used. In general We have used the inhibitor in final concentrations- (in the blood) varying between 0.02

to-0.03 molar. 1

a In the appended tables are set forth representative results-of our investigations demonstrating the'effectiveness of various soluble salts of salicylic acid and acetyl salicylic acid as leucolysis inhibitors at varying temperatures that may be encountered throughoutthe year and a comparison of results with blood specimens to which no inhibitor has been added.

TABLE ,I

Leucocyte counts of stored human blood with and without leucolysis inhibitor itor on erythrocyte and hemoglobin content of human blood Leucolysis inhib- Leucocytes percubic millimeter Fahrenheit itor used of blood after:

Tempera: Specimen No. ture of Stored Amount Blood mud in Moles 10 Min. 24 hrs. 48 hrs. 72l1rs.

p per liter- 1 None" 7, 200 6, 500 5, 600 4, 000 2 -85 None 10, 200 5, 800 2 600 98.6 None. 10,200 4,900 4 A 0. 01 6, 300 6, 500 6, 000 5 40 A 0.02 7, 000 7, 200 7, 000 6, 800 6880 B 0.02 10,400 10,200 10, 200 10, 000 98.6.. A 0.04 9,900 9,700 8,800 8 -88 B 0.02 87, 500 87, 000 88, 000 87, 600 9 98.6.... A 0.02 5, 700 5,800 5 200 10 5 hrs. B 0. 02 9, 8, 900 8,500

113 then 73-83 l1- 7080 C 0. 01 10, 500 10, 500 10, 100 12 98L6 O 0.02 6,800 6, 600 13- 6-- D 0. 02 12, 900 12, 800

1 A designates sod iumsalt oi ortho hydroxybenzoic acid. I B designates lithium salt of ortho hydroxybenzoic acid. 5 O designates potassium salt of ortho hydroxybenzoic acid. 4 D designates potassium salt of ortho acetoxybenzoic acid.

TABLE II Showing absence of influence of leucolysis inhib- Erythrocyte and Hemoglobin content after- Fahrem Concentrai he: lnlnbtion of Specimen No. 1tor Inhibitor in 10 min. 24 hrs. 48 hrs. 72 hrs.

pew used Mollejs per 1 er E H E H E H E H 98. 6 C 3 0.02 5. 3 14. 2 5. 1 14. 2 D 4 0.01 4.3 12.5 4.2 1216 A 5 0. 02 4.3 12.3 4.4 12.4 4.3 12.5 4.4 12.5 A 5 0. 02 5. 6 16.1 5. 7 15. 8 5. 6' 15. B 6 0.02 4.0 12.0 4:1 12.2 3.9 12.0 3.9 12.1 B B 0.02 5.1 15.5 5. 2 15.3

1 E designates erythrocytes, millions per cubic millimeter. 1 H designates hemoglobin, grams per 100 cc.

' O designates potassium salt of orthy hydroxyhenzoic acid.

4 D designates potassium salt of ortho acetoxybenzoic acid. 5 A designates sodium salt of orthy hydroxybenzoic acid. 6 B designateslithium' salt 'of ortho hydroxybenzoic acid.

It willbe understood that the freshly drawn blood. may be treated with the anticoagulant and the leucolysis inhibitors by the addition thereto of a, requisite quantity of a solution containing both the anticoagulant and leucolysis inhibitor or .vated as 113 F. without in any way aifecting its usefulness for the making of an accurate blood count.

It will be obvious that it is possible to modify the procedures hereinabove described for treating blood with the leucolysis inhibitors. Accordingly, variations in the technique of treating blood, or the separated leucocytes, so as to preserve the same and inhibit leucolysis are capable of accomplishment without departing from the spirit of our invention.

The appended claims therefore are to be understood as defining the invention within the full spirit and scope thereof.

We claim:

, 1. Preserved blood comprising: blood; an anticoagulant; and at least a 0.01 molar concentration of a water-soluble salt of a member of the group consisting of salicylic acid and acetyl salicyclic acid.

2. Preserved blood comprising: blood; an anticoagulant; and at least a 0.01 molar concentration of an alkali metal salt of a member of the group consisting of salicylic acid and acetyl salicylic acid.

3. Preserved blood comprising: blood; an anticoagulant; and at least a 0.01 molar concentration of a potassium salt of a member of the group consisting of salicylic acid and acetyl salicylic acid.

4. Preserved blood comprising: blood; an anticoagulant; and at least a 0.01 molar concentration of a sodium salt of a member of the group consisting of salicylic acid and acetyl salicylic acid.

5. Preserved blood comprising: blood; an anticoagulant; and at least a 0.01 molar concentra tion of a lithium salt of a member of the group consisting of salicylic acid and acetyl salicylic acid.

6. A container for the transport of blood used for determining the hemoglobin, erythrocyte and leucocyte content of blood which comprises: a specimen tube containing an adherent deposition of an anticoagulant and a water-soluble salt of a member of the group consisting of salicylic acid and acetyl salicylic acid, said salt in an amount, in relation to the volume of the bloodspecimen to be contained in said container, which will produce at least about a 0.01 molar concentration of the aforesaid salt in the blood-specimen to be contained in said container.

7. A container for the transport of blood used for determining the hemoglobin, erythrocyte and leucocyte content of blood which comprises: a specimen tube containing an adherent deposition of an anticoagulant and an alkali metal salt of a member of the group consisting of salicylic acid and acetyl salicylic acid, said salt in an amount, in relation to the volume of the blood-specimen to be contained in said container, which will produce at least about a 0.01 molar, concentration of the aforesaid salt in the blood-specimento be contained in said container.

8. A container for the transport of blood used for determining the hemoglobin, erythrocyte and leucocyte content of blood which comprises: a specimen tube containing an adherent deposition of an anticoagulant and a potassium salt of a member of the group consisting of salicylic acid and acetyl salicylic acid, said salt in an amount, in relation to the volume of the blood-specimen to be contained in said container, which will produce at least about a 0.01 molar concentration of the aforesaid salt in the blood-specimen to be contained in said container.

9. A container for the transport of blood used for determining the hemoglobin, erythrocyte and leucocyte content of blood whichcomprises: a specimen tube containing an adherent deposition of an anticoagulant and a sodium salt of a member of the group consisting of salicylic acid and acetyl salicylic acid, said salt in an amount, in relation to the volume of the blood-specimento be contained in said container, which will produce at least about a 0.01 molar concentration of the aforesaid salt in the blood-specimen to becontained in said container.

10. A container for the transport of blood used for determining the hemoglobin, erythrocyte and leucocyte content of blood which comprises; a specimen tube containing an adherent deposition of an anticoagulant and a lithium salt of a -member of the group consisting of salicylic acid and acetyl salicylic acid, said salt in an amount, in relation to the volume of the blood-specimen to be contained in said container, which will produce at least about a 0.01 molar concentration of the aforesaid salt in the blood-specimen to be contained in said container.

11. An article of manufacture, for the preparation and transportation of a blood-specimen to be used for the delayed determination of the hemoglobin, erythrocyte and leucocyte content thereof, comprising a specimen-container adapted to receive a, predetermined volume of freshlydrawn blood and to retain the same in a sealed condition, said container containing an anti-coagulant together with a water-soluble salt of a member of the group consisting of salicylic acid and acetyl salicylic acid, the said salt in an amount, in relation to the volume of the bloodspecimen to be contained in said specimen-con tainer, which will produce between about a, 0.01 and 0.08 molar concentration of the aforesaid salt in the blood-specimen to be contained and sealed in said container.

12. An article of manufacture, for the preparation and transportation of a blood-specimen to be used for the delayed determination of the hemoglobin, erythrocyte and leucocyte content thereof, comprising a specimen-container adapted to receive a predetermined volume of freshlydrawn blood and to retain the same in a, sealed condition, said container containing an anticoagulant together with an alkali metal salt of a member of the group consisting of salicylic acid and acetyl salicylic acid, the said salt in an amount, in relation to the volume of the blood-specimen to be contained in said specimencontainer, which will produce between about a 0.01 and 0.08 molar concentration of the aforesaid salt in the blood-specimen to be contained and sealed in said container.

13. An article of manufacture, for the preparation and transportation of a blood-specimen to be- 11sec for the delayed determination of the i hemoglobin, erythrocyte and leucocyte content thereof, comprising a specimen-container adapted to receive a predetermined volume of freshlydrawnbl'ood and to'retain the same in a sealed condition, saidcontainer containing an anticoagulant together with a potassium salt ofa member of the group consistin of salicylic acid and acetylsalicylicacid, the said: salt in an mount, in relation to the volume of the bloodspecimen to be containedin said specimen-container, which will produce between about a 0.01 and 0.08' molar concentration of the aforesaid the group consisting of salicylic acid and acetylsalicylic'acid; the said salt in an amount, in relation to the volume of the blood-specimen to be contained in saidspecimen-container, which will produce between about a-0.0 1 and 0.08 molar concentration of the aforesaid salt in the bloodspecimento be contained and sealed in said contamer. V

15. article of manufacture, for the prepsalt in the blo'od-specimen to be contained and aration. and transpor-tation of a. blood-specimen to be used for the delayed determinationof the hemoglobin, erythrocyte and. leucocyte: content thereof; comprising a specimen containeradapt= ed'to receive a predeterminedvolume of. freshly drawn blood and to retain. the same in a sealed condition, said container containing an" anti.- coagulant together with a lithium salt of ia member of the group consisting of salicylic acid and acetylsalicylic acid; the said. saltin an amount, in relation to the-volume of the bl'oo'd specimen to be contained in said specimen-container, which will produce between about a- 0:01 and 0.08 molar concentration. of theaforesaid salt in the blood-specimen to be contained and sealed in said container.

HERMAN BROWNi MEYER SAMSON? REFERENGES (BIIED The following references are of record in' the file of this patent: 

1. PRESERVED BLOOD COMPRISING: BLOOD; AN ANTICOAGULANT; AND AT LEAST A 0.01 MOLAR CONCENTRATION OF A WATER-SOLUBLE SALT OF A MEMBER OF THE GROUP CONSISTING OF SALICYLIC ACID AND ACETYLE SALICYCLIC ACID. 